This invention relates to methods for screening compound libraries, such as compound libraries generated using combinatorial chemistry techniques. The methods of this invention employ frontal chromatography in combination with mass spectrometry to screen a library of compounds to identify and rank those members of the library that bind to a target receptor. The methods of this invention also permit a compound library to be rapidly screened to determine if one or more members of the library have an affinity for a target receptor as measured by a pre-selected indicator compound.
The following publications, patents and patent applications are cited in this application as superscript numbers:
1 K. S. Lam, Anti-Cancer Drug Des. 1997, 12, 145-167.
2 P. M. Sweetnam et al., In Burger""s Medicinal Chemistry and Drug Discovery; M. E. Wolff, Ed.; John Wiley and Sons: New York, 1995; pp 697-731.
3 R. H. Griffey et al., In Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, Calif., June 1-5, 1997; p. 400.
4 L. Fang et al., In Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, Calif., Jun. 1-5, 1997; p. 401.
5 Y.-H. Chu et al., J. Am. Chem. Soc. 1996, 118, 7827-7835.
6 Y.-Z. Zhao et al., J. Med. Chem. 1997, 40, 4006-4012.
7 Y. F. Hsieh et al., J. Mol. Div. 1996, 2, 189-196.
8 R. W. Nelsonet al., Anal. Chem. 1995, 67, 1153-1158.
9 D. C. Schriemer and L. Li, Anal. Chem. 1996, 68, 3382-3387.
10 PCT/US97/07964 (International Publication No. WO 97/43641), published Nov. 20, 1997, entitled xe2x80x9cMolecular Diversity Screening Device and Method.xe2x80x9d
11 R. Wieboldt et al., Anal. Chem. 1997, 69, 1683-1691.
12 R. B. van Breemen et al., Anal. Chem. 1997, 69, 2159-2164.
13 M. L. Nedved et al., Anal. Chem. 1996, 68, 4228-4236.
14 PCT/US95/03355 (International Publication No. WO 95/25737), published Sep. 28, 1995, entitled xe2x80x9cMethod for Identifying Members of Combinatorial Libraries.xe2x80x9d
15 PCT/EP97/02215 (International Publication No. WO 97/43301), published Nov. 20, 1997, entitled xe2x80x9cIdentification of Members of Combinatorial Libraries By Mass Spectrometry.xe2x80x9d
All of the above publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
In recent years, a large number of combinatorial chemistry techniques have been developed which permit vast libraries of diverse chemical compounds to be rapidly synthesized.1 In combinatorial chemistry, a series of chemical reactions is typically conducted employing a plurality of reagents at each step to generate a library of compounds. Such techniques have the potential to greatly accelerate the discovery of new compounds having biologically useful properties by providing large collections of diverse chemical compounds for biological screening.
This ability to rapidly generate large collections of compounds using combinatorial chemistry techniques has created a need for new methods of screening compound libraries. The traditional approach of screening each compound individually in an assay to identify those compounds having the desired biological activity is no longer practical due to time and resource constraints. Thus, a need exists for new methods which permit the rapid screening of compound libraries.
In this regard, various methods for screening compound libraries have been reported. Typically, these screening methods involve the use of target receptors which have been labeled with fluorescent or other reporter groups.2 In these methods, the compound library, typically bound to a resin bead, is exposed to the labeled target receptor and those members binding to the labeled target receptor are identified and physically separated. The particular ligand binding to the target receptor is then identified. In many of these techniques, elaborate procedures are required to keep track of individual members of the library. For example, coded tags are often added during the synthesis of the combinatorial library to allow the structure of the individual members to be subsequently determined. Alternatively, combinatorial libraries can be prepared in an array and the individual members of the library subsequently identified by their location in the array. While such methods can be effective, the need to keep track of individual members of the library during their synthesis and screening is quite cumbersome and often limits the type of synthetic procedures that can be employed. Additionally, many of these techniques require that the synthetic procedures be conducted on a solid phase, thus further limiting the synthetic procedures and reagents that can be used.
As an alternative, mass spectrometry is emerging as an important tool for the interrogation of combinatorial libraries. To date, mass spectrometry has been used to assess library quality3,4 and, when coupled with molecular recognition technologies, has allowed for some success in the isolation and characterization of active library compounds.5-15 Typically, when screening compound libraries for biologically active members, mass spectrometry is used in combination with a xe2x80x9ccapture and releasexe2x80x9d methodology. In this methodology, compound mixtures are presented to the target receptor, which is often immobilized on a solid support, and the resulting ligand-receptor complexes are separated from the unbound members of the library. After separation, the ligand-receptor complexes are typically denatured, for example, with a solvent and the solvent mixture containing the previously bound ligands is presented to the mass spectrometer to permit identification of the high affinity ligands.
For example, ultrafiltration has been used in combination with electrospray mass spectrometry to screen combinatorial libraries.10-12 In this method, ligands present in a compound library are allowed to bind to a receptor and the resulting ligand-receptor complexes are purified by ultrafiltration. The ligand-receptor complexes are then dissociated using a solvent, such as methanol, and the previously bound ligands are detected by an electrospray mass spectrometer.
Affinity capillary electrophoresis (ACE) has also been coupled with mass spectrometry to screen combinatorial libraries.5 In this procedure, ACE is used to separate ligand-receptor complexes from unbound ligands and mass spectrometry is used to identify the high affinity ligands.
Similarly, compound libraries have been screened using affinity chromatography in combination with mass spectrometry. For example, WO 97/43301 describes a method for characterizing the members of a combinatorial library, which method utilizes affinity selection in combination with mass spectrometry. Specifically, the members of the library are brought into contact with a domain of interest to allow for binding, i.e., the formation of a complex. After binding, the complex is separated from the unbound members of the library, typically by washing the unbound members from the column containing the complexes. The complexes are then treated to elute the bound library components and the eluted components are analyzed by mass spectrometry. The elution methods described include the use of displacers, chaotrope agents, pH elution, salt gradients, temperature gradients, organic solvents, selective denaturants and detergents. Using such methods, the weakly bound members of the library are purportedly eluted first and analyzed by mass spectrometry, followed by the elution of the more strongly bound members.
There are several disadvantages associated with the: xe2x80x9ccapture and releasexe2x80x9d methods for screening compound libraries that have been previously reported. First, the procedure used to xe2x80x9creleasexe2x80x9d the bound ligands from the ligand-receptor complexes may alter the binding profile for the various bound ligands, resulting in a false indication of binding strength. For example, using a pH gradient to release the bound members of the library may change the electronic character of the binding site on the receptor causing ligands which are strongly bound under physiological conditions to be prematurely released. Thus, the characterization of binding strength for various ligands based on their relative time of release may be misleading if the release conditions are different from the binding conditions. Accordingly, these methods may not accurately identify the most active members of a compound library. Additionally, certain conditions used for compound release, such as pH gradients, may irreversibly denature the receptor thus preventing its subsequent use for screening compound libraries.
Additionally, when xe2x80x9ccapture and releasexe2x80x9d methods are employed, each bound ligand is typically released over a relatively short period of time resulting, for example, in an elution peak or xe2x80x9cspikexe2x80x9d for each ligand. Accordingly, the effluent produced using such methods is typically monitored continually, for example, by mass spectrometry so that any particular elution peak is not missed. Thus, the number of analyses that can be conducted using any particular mass spectrometer is limited. Accordingly, it would be desirable to develop methods for screening compound libraries that do not rely upon xe2x80x9ccapture and releasexe2x80x9d methodologies.
This invention is directed to methods for screening compound libraries. The compound libraries may be generated or obtained by any means including, by way of example, combinatorial chemistry techniques or from fermentation broths, plant extracts, cellular extracts and the like. The methods of this invention employ frontal chromatography (FC) in combination with mass spectrometry (MS) to screen the library of compounds to identify and rank those members of the library that bind to a target receptor.
In frontal chromatography, a target receptor is typically immobilized on a suitable solid support material and packed in a column. A mixture containing putative ligands is then continuously infused through the column. Ligands having an affinity for the target receptor bind to the column, but eventually the capacity of the column for each ligand is exceeded and the ligands elute or xe2x80x9cbreak throughxe2x80x9d at their infusion concentration. Once a ligand begins eluting from the column, it is continually present in the effluent. Compounds having little or no affinity for the target receptor break through earlier in the effluent compared to ligands having a higher affinity for the receptor.
In the present invention, mass spectrometry (MS) is employed to continuously or intermittently monitor the FC effluent. Using MS, the identity and break through time for each ligand on the column can be determined. Thus, FC-MS allows the relative affinity of each member of the library for the target receptor to be determined relative to other members of the library under ligand-receptor binding conditions. Using the present methods, an accurate ranking of the relative affinity of each member of the compound library for the target receptor can be ascertained.
Accordingly, in one of its method aspects, the present invention is directed to a method for screening a compound library to determine the relative affinity of a plurality of putative ligands to a target receptor or a plurality of target receptors, which method comprises:
(a) providing a compound library comprising a plurality of putative ligands,
(b) applying the compound library to a column comprising a target receptor or a plurality of target receptors, each target receptor optionally bound to a solid phase support, under frontal chromatography conditions to provide an effluent;
(c) continuously or intermittently applying the effluent to a mass spectrometer to provide mass spectra of the constituent putative ligands present in the effluent; and
(d) evaluating the mass spectra to determine a break through time for the putative ligands.
In a preferred embodiment, the above method further comprises the step of:
(e) determining an affinity to the target receptor for a putative ligand in the compound library relative to another putative ligand in the library by comparing the break through time for the putative ligand to the break through time for the other putative ligand.
In another preferred embodiment, the above method further comprises the step of:
(f) determining a dissociation constant, Kd, for a putative ligand in the compound library and the target receptor.
Preferably, the compound library employed in the above method comprises less than about 10,000 putative ligands, more preferably less than about 5,000 putative ligands. Still more preferably, the compound library comprises about 5 to about 100 putative ligands.
In one embodiment of this invention, the compound library preferably comprises putative ligands selected from the group consisting of carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleosides, nucleotides, oligonucleotides, polynucleotides, lipids, retinoids, steroids, glycopeptides, glycoproteins, glycoproteins, proteoglycans, and synthetic analogs or derivatives thereof, and the like.
In another embodiment, the compound library comprises putative ligands selected from the group consisting of natural products.
In another embodiment, the compound library comprises putative ligands selected from the group consisting of synthetic small molecule organic compounds.
Preferably, each target receptor is independently selected from the group consisting of proteins, including recombinant proteins, glycoproteins, glycosaminoglycans, proteoglycans, integrins, enzymes, lectins, selectins, cell-adhesion molecules, toxins, bacterial pili, transport proteins, receptors involved in signal transduction or hormone-binding, hormones, antibodies, major histocompatability complexes, immunoglobulin superfamilies, cadherins, DNA or DNA fragments, RNA and RNA fragments, whole cells, cell fragments, tissues, bacteria, fungi, viruses, parasites, preons, and synthetic analogs or derivatives thereof.
Additionally, the target receptor is preferably bound to a solid phase support. More preferably, the target receptor is covalently bound to the solid phase support or bound via biotin-avidin or biotin-streptavidin binding.
Preferably, the solid phase support used in this invention is selected from the group consisting of polymeric (resin) beads, polymeric gels, glass beads, silica chips, silica capillaries, agarose, diatomaceous earths and pulp.
The column employed in the methods of this invention preferably contains from about 1 fmol to about 10 nmol of target receptor active sites; preferably, from about 1 pmol to about 10 nmol of target receptor active sites.
In the methods of this invention, the effluent from the column is preferably diluted with a supplemental diluent before analysis by mass spectrometry. Preferably, the supplement diluent comprises a major amount of an organic solvent and a minor amount of an aqueous buffer. The organic solvent is preferably selected from the group consisting of acetonitrile, methanol and isopropanol.
Preferably, the mass spectrometer employed in the methods of this invention is an electrospray mass spectrometer.
Additionally, since FC-MS does not require constant effluent monitoring, a plurality of FC-MS analyses can be conducted simultaneously using a single mass spectrometer to intermittently monitor each column. Furthermore, under FC conditions, since ligands are always present in the effluent once they break through the column, the intermittent monitoring of each column does not necessarily require monitoring the actual break through time for each ligand. Therefore, a plurality of FC-MS analyses can be conducted simultaneously using a single mass spectrometer to intermittently monitor each column.
Accordingly, in another of its method aspects, this invention provides a method for screening a plurality of compound libraries to determine the relative affinity of a plurality of putative ligands in each library to a target receptor, which method comprises:
(a) providing a plurality of compound libraries, each library comprising a plurality of putative ligands,
(b) applying each compound library to a separate column comprising a target receptor or a plurality of target receptors, each target receptor optionally bound to a solid phase support, under frontal chromatography conditions to provide an effluent from each column;
(c) intermittently applying the effluent from each column to a mass analyzer of a mass spectrometer to provide mass spectra of the constituent putative ligands present in the effluent; and
(d) evaluating the mass spectra to determine a break through time for the putative ligands in each compound library.
In a preferred embodiment, the above method further comprises the step of:
(e) determining an affinity to the target receptor for a putative ligand in a compound library relative to another putative ligand in the same library by comparing the break through time for the putative ligand to the break through time for the other putative ligand.
Preferably, from 2 to about 100 columns are used in the methods of this invention which employ a plurality of columns. More preferably, 2 to about 50 columns are employed and still more preferably, 2 to about 10 columns are employed.
In another embodiment, this invention provides a method for screening a compound library to determine if putative ligands of the library have an affinity for a target receptor as measured by a pre-selected indicator compound having known affinity for the target receptor. Using this embodiment, compound libraries can be rapidly screened to identify those libraries that contain ligands capable of shifting the break through time of the pre-selected indicator compound. This shift in break through time for the indicator compound, which can be either a positive time shift or a negative time shift, indicates that putative ligands in the library have an affinity for the target receptor. Optionally, more than one indicator compound can be used for each target receptor or target receptor active site.
Accordingly, in another of its method aspects, this invention provides a method for screening a compound library to determine the relative affinity of a plurality of putative ligands to a target receptor or a plurality of target receptors relative to an indicator compound or a plurality of indicator compounds, which method comprises:
(a) providing a compound library comprising a plurality of putative ligands;
(b) providing at least one void marker compound;
(c) providing an indicator compound or a plurality of indicator compounds for each target receptor, each indicator compound having a pre-determined affinity for the target receptor and having a pre-determined break through time on the column in the absence of the compound library relative to a void marker compound;
(d) applying the compound library to a column comprising a target receptor or a plurality of target receptors, each target receptor optionally bound to a solid phase support, under frontal chromatography conditions to equilibrate or partially equilibrate the column with the compound library;
(e) applying (i) a mixture comprising the compound library, the void marker compound and the indicator compound or compounds, or (ii) the void marker compound and the indicator compound or compounds, to the column under frontal chromatography to provide an effluent;
(f) analyzing the effluent to determine a break through time for the indicator compound or compounds.
In a preferred embodiment, the above method further comprises the step of:
(g) determining whether any putative ligands of the compound library have an affinity for the target receptor as measured by the indicator compound or compounds by comparing the break through time for the indicator compound or compounds from step (f) with the pre-determined break through time for the indicator compound or compounds in the absence of the compound library.
Preferably, the analysis of the effluent in the above method is conducted using a mass spectrometer.
Preferably, when an indicator compound is used, the compound library comprises less than about 50,000 putative ligands, more preferably less than 10,000 putative ligands. Still more preferably, the compound library comprises about 5 to about 100 putative ligands.
Preferably, the indicator compound employed in the methods of this invention has a pre-determined break through time in the absence of the compound library of less than about 5 minutes.
A plurality of columns may also be employed when an indicator compound is used. Accordingly, in still another of its method aspects, the present invention provides a method for screening a plurality of compound libraries to determine the relative affinity of a plurality of putative ligands to a target receptor or a plurality of target receptors relative to an indicator compound, which method comprises:
(a) providing a plurality of compound libraries comprising a plurality of putative ligands;
(b) providing at least one void marker compound;
(c) providing an indicator compound or a plurality of indicator compounds for each target receptor, each indicator compound having a pre-determined affinity for the target receptor and having a pre-determined break through time on each column in the absence of the compound library relative to a void marker compound;
(d) applying each compound library to a separate column comprising a target receptor or a plurality of target receptors, each target receptor optionally bound to a solid phase support, under frontal chromatography conditions to equilibrate or partially equilibrate the column with the compound library;
(e) applying (i) a mixture comprising the compound library, the void marker and the indicator compound or compounds, or (ii) the void marker and the indicator compound or compounds, to each column under frontal chromatography conditions to provide an effluent;
(f) analyzing the effluent from each column to determine a break through time for the indicator compound or compounds.
In a preferred embodiment, the above method further comprises the step of:
(g) determining whether any putative ligands of a compound library have an affinity for the target receptor as measured by the indicator compound or compounds by comparing the break through time for the indicator compound or compounds from step (f) with the pre-determined break through time for the indicator compound or compounds in the absence of the compound library.
An indicator compound can also be used to determine if the overall affinity of a compound library for a target receptor is due to a plurality of weak binders or to one or more strong binders present in the compound library. Accordingly, in yet another of its method aspects, the present invention provides a method for screening a compound library to determine the relative affinity of a plurality of putative ligands to a target receptor relative to an indicator compound having a pre-determined affinity for the target receptor, which method comprises:
(a) providing a compound library comprising a plurality of putative ligands;
(b) providing at least one void marker compound;
(c) providing a column comprising a target receptor optionally bound to a solid phase support;
(d) providing an indicator compound having a pre-determined affinity for the target receptor and having a pre-determined break through time on the column in the absence of the compound library relative to a void marker compound and having a pre-determined signal intensity in the presence of the compound library;
(e) applying a mixture comprising the compound library and the indicator compound to the column under frontal chromatography conditions to provide an effluent;
(f) analyzing the effluent to determine a break through time and/or signal intensity for the indicator compound.
In a preferred embodiment, the above method further comprises the step of:
(g) determining whether any putative ligands of the compound library have an affinity for a target receptor as measured by the indicator compound by comparing the break through time for the indicator compound from step (f) with the pre-determined break through time for the indicator compound in the absence of the compound library.
In another preferred embodiment, the above method further comprises the step of:
(h) determining whether the affinity for the target receptor is due to a plurality of ligands having weaker affinity for the target receptor relative to the indicator compound or to one or more ligands having stronger affinity for the target receptor relative to the indicator compound by comparing the signal intensity of the indicator in the effluent with the predetermined signal intensity for the indicator.
The methods of the present invention also allow the loss of function of a target receptor, such as an enzyme, to be monitored in the presence of a compound library. Using this embodiment, compound libraries containing inhibitors of a target receptor can be readily identified. Accordingly, in another of its method aspects, this invention provides a method for screening a compound library to identify inhibitors of a target receptor, which method comprises:
(a) providing a compound library comprising a plurality of putative ligands,
(b) applying the compound library to a column comprising a target receptor optionally bound to a solid phase support under frontal chromatography conditions;
(c) providing a first indicator compound which is capable of being chemically modified by the target receptor to form a second indicator compound;
(d) applying (i) a mixture comprising the compound library and the first indicator compound, or (ii) the first indicator compound, to the column under frontal chromatography conditions to provide an effluent;
(e) analyzing the effluent to determine the presence and/or concentration of the first indicator compound and/or the second indicator compound.
Preferably, the target receptor employed in the above method is a catalyst and the first indicator compound is a reactant for the catalyst. More preferably, the target receptor is an enzyme and the first indicator compound is a substrate for the enzyme.
The methods of this invention can also be employed to screen a target receptor or a plurality of target receptors for affinity to an immobilized ligand or plurality of ligands. This embodiment is particularly useful for target validation studies on ligands having biological effects. Accordingly, in another of its method aspects, this invention provides a method for screening a target receptor or a plurality of target receptors to determine the relative affinity of the receptor or receptors to an immobilized ligand or ligands relative to an indicator compound or a plurality of indicator compounds, which method comprises:
(a) providing a target receptor or a plurality of target receptors;
(b) providing a column comprising a ligand or a plurality of ligands, each ligand bound to a solid phase support;
(c) providing at least one void marker compound;
(d) providing an indicator compound or a plurality of indicator compounds for each ligand, each indicator compound having a pre-determined affinity for the ligand and having a pre-determined break through time on the column relative to a void marker compound;
(e) applying the target receptor or receptors to the column under frontal chromatography conditions to equilibrate or partially equilibrate the column with the target receptor or receptors;
(f) applying (i) a mixture comprising the target receptor or receptors, the void marker compound and the indicator compound or compounds, or (ii) the void marker compound and the indicator compound or compounds, to the column under frontal chromatography conditions to provide an effluent;
(g) analyzing the effluent to determine a break through time for the indicator compound or compounds.
In a preferred embodiment, the above method further comprises the step of:
(h) determining whether any of the target receptors has an affinity for the ligand or ligands as measured by the indicator compound or compounds by comparing the break through time for the indicator compound or compounds from step (g) with the pre-determined break through time for the indicator compound or compounds.
This method can also be used to screen a compound libraries comprising a plurality of putative ligands immobilized on a solid phase support for affinity to a target receptor or a plurality of target receptors.
In the methods of this invention, the effluent from the column can be introduced directly into the mass spectrometer or the effluent can be collected in fractions and the fractions subsequently analyzed by mass spectrometry. In either embodiment, the effluent can optionally be pre-treated, such as by desalting or by derivatizing the components, prior to analysis. Accordingly, in one preferred embodiment, each of the above methods optionally comprise the steps of collecting the effluent from column to provide a plurality of effluent fractions; and optionally desalting each effluent fraction prior to mass spectral analysis.